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Revista Brasileira de Farmacognosia

Print version ISSN 0102-695XOn-line version ISSN 1981-528X

Rev. bras. farmacogn. vol.28 no.2 Curitiba Mar./Apr. 2018

https://doi.org/10.1016/j.bjp.2018.01.006 

Original articles

Comparison of bioactive compounds content in leaf extracts of Passiflora incarnata, P. caerulea and P. alata and in vitro cytotoxic potential on leukemia cell lines

Marcin Ozarowskia  b  * 

Anna Piaseckac  d 

Anna Paszel-Jaworskae 

Douglas Siqueira de A. Chavesf 

Aleksandra Romaniuke 

Maria Rybczynskae 

Agnieszka Gryszczynskab 

Aneta Sawikowskag  h 

Piotr Kachlickic 

Przemyslaw L. Mikolajczakb  i 

Agnieszka Seremak-Mrozikiewiczb  j  k 

Andrzej Klejewskim 

Barbara Thiema 

aDepartment of Pharmaceutical Botany and Plant Biotechnology, Poznan University of Medical Sciences, Poznan, Poland

bDepartment of Pharmacology and Phytochemistry, Institute of Natural Fibers and Medicinal Plants, Poznan, Poland

cDepartment of Pathogen Genetics and Plant Resistance, Metabolomics Team, Institute of Plant Genetics of the Polish Academy of Science, Poznan, Poland

dInstitute of Bioorganic Chemistry of the Polish Academy of Science, Poznan, Poland

eDepartment of Clinical Chemistry and Molecular Diagnostics, Poznan University of Medical Sciences, Poznan, Poland

fDepartment of Pharmaceutical Science, Health and Biological Science Institute, Federal Rural University of Rio de Janeiro, Seropédica, RJ, Brazil

gDepartment of Biometry and Bioinformatics, Institute of Plant Genetics, Polish Academy of Science, Poznan, Poland

hDepartment of Mathematical and Statistical Methods, Poznań University of Life Sciences, Poznań, Poland

iDepartment of Pharmacology, Poznan University of Medical Sciences, Poznan, Poland

jDivision of Perinatology and Women's Diseases, Poznan University of Medical Sciences, Poznan, Poland

kLaboratory of Molecular Biology, Poznan University of Medical Sciences, Poznan, Poland

mDepartment of Nursing, University of Medical Sciences, Poznan, Poland


ABSTRACT

Passiflora caerulea L., P. alata Curtis and P. incarnata L. (synonym for P. edulis Sims), are the most popular representatives of the Passiflora genus in South America. In recent years, a growing attention is paid to the biological activity and phytochemical profiles of crude extracts from various species of Passiflora in worldwide. The aim of this study was to evaluate and to compare of anti-leukemic activity of the dry crude extracts from leaves of three Passiflora species from greenhouse of Poland in two human acute lymphoblastic leukemia cell lines: CCRF-CEM and its multidrug resistant variant. Two systems of liquid chromatography in order to assessment of phytochemical composition of extracts were applied. Extracts of P. alata and P. incarnata showed the potent inhibitory activity against human acute lymphoblastic leukemia CCRF-CEM, while P. caerulea not showed activity (or activity was poor). Despite similarities in quality phytochemical profile of extracts from P. caerulea and P. incarnata, differences in quantity of chemical compounds may determine their various pharmacological potency. For the activity of P. alata extract the highest content of terpenoids and a lack of flavones C-glycosides are believed to be crucial. Summarizing, the crude extract from P. alata leaves may be considered as a substance for complementary therapy for cancer patients.

Keywords: Passion flower; HPLC-ESI-MSn/UPLC-PDA; Flavones C-glycosides; Terpenoids; Anti-leukemic activity

Introduction

Passiflora incarnata L., P. caerulea L. and P. alata Curtis belong to the family Passifloraceae, consisting of eighteen genera and approximately 630 species (Perez et al., 2007). Passiflora plants are used in traditional medicine not only in South America (Dhawan et al., 2004), but also in the Netherlands, Spain, Italy and Poland (Ozarowski and Thiem, 2013). They exhibit various pharmacological activities and possess complex, biologically active compounds (Miroddi et al., 2013). P. incarnata (purple passion flower), originating from North America, is one of the most important medicinal plants, and is a source of valuable herbal substance (Passiflorae herba) described in European Pharmacopoeia and by European Medicines Agency (EMA, 2014). According to the international plant list, P. incarnata is a synonym of P. edulis Sims. P. edulis is scientific name accepted (The Plant List, 2017). This herb has been commonly used in traditional phytotherapy in sleep disorders, nervousness, and anxiety for a long time throughout the world (Miroddi et al., 2013). Passiflora alata (winged-stem passion flower), native to the Amazon, from Peru to eastern Brazil, is officially recognized as a medicinal plant described in the Brazilian Pharmacopoeia (Farmacopeia Brasileira, 2010). Besides of P. alata, P. edulis is also in the Brazilian Pharmacopoeia. The edible fruits of this species are economically important for food industry. Passiflora caerulea (blue passion flower), native to Brazil, is possibly the most known species of the Passifloraceae family, but, to date, there are any data on the complete phytochemical profile of the extract from P. caerulea leaves. The first time the plant material from P. caerulea has been recognized as equivalent to the P. incarnata by Oswiecimska (1956).

Recent study demonstrated that the ethanolic extract of P. caerulea showed anti-inflammatory, anti-diarrhoeal and spasmolytic activities in experimental colitis model (Anzoise et al., 2016). Moreover, it was observed that a flavone chrysin (from P. caerulea) increased libido and sperm count in rats (Dhawan et al., 2002).

In the recent years, a growing attention is paid to the biological activity and phytochemical profiles of extracts from different species of Passiflora genus (Pereira et al., 2004; Ozarowski and Thiem, 2013; Farag et al., 2016; Wosch et al., 2017). Moreover, very interesting is chemical composition of leaf extracts from plants growing in greenhouse controlled conditions in polish terms and the relationship between the presence of active compounds in the extracts of P. incarnata, P. alata and P. caerulea and their anti-leukemic activity.

To the best of our knowledge, the crude extracts from leaves of P. incarnata, P. alata and P. caerulea were not tested on the acute human leukemia cell lines. Acute leukemias are malignant clonal disorders of hematopoietic precursor cells, in which leukemic stem cells acquire mutations that confer self-renewal capabilities, altered hematopoietic differentiation, and increased proliferative capacity (Zhou et al., 2013). Therefore, in vitro screening of plant extracts containing various chemical compounds may essentially contribute to the discovery and development of new, clinically useful drugs of natural origin as an alternative strategy for prevention and management of leukemia.

Materials and methods

Plant material

Leaves of Passiflora incarnata L., P. alata Curtis, P. caerulea L., Passifloraceae, were obtained from plants grown in the greenhouse of Department of Medicinal and Cosmetic Natural Products, University of Medical Sciences, Poznan. Controlled condition of greenhouse as follows: temperature range from 25 ºC to 40 ºC, 60–70% humidity. The plants grew on a substrate composed of peat: gravel (3:1) plus Substral Osmocote® Universal.

The material was identified at the Department of Medicinal and Cosmetic Natural Products, Faculty of Pharmacy, Poznan University of Medical Sciences. The voucher specimens (no. 15.174, no. 15.175, no. 15.176 respectively) have been deposited in the Herbarium of the Institute of Natural Fibers and Medicinal Plants in Poznan, Poland.

Chemicals and reagents

Adriamycin and MTT solution were obtained from Sigma–Aldrich (St. Louis, MO, USA). Solvents for extraction and LC–MS analyses (methanol, acetonitrile, formic acid and ultrapure water) were obtained from Sigma–Aldrich (Poznan, Poland).

Preparation of the extracts

Dry leaves (5 g) after drying oven with air circulation (25 ºC, 24 h) were extracted with methanol pure PA (1:10, m/V) three times for 1 h by reflux. Next, the extract was concentrated under vacuum to eliminate the methanol content. The yields of the dry extracts were 32.6% for P. caerulea, 27.9% for P. alata, 21.9% for P. incarnata.

Metabolite identification with LC–MS systems

Identification of secondary metabolites presented in the extracts of leaves from Passiflora species were performed using two complementary LC–MS systems. The first system HPLC-DAD-MSn consisted of Agilent 1100 HPLC instrument with a photodiode-array detector PDA (Palo Alto, CA, USA) and Esquire 3000 ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) with the X Bridge C18 column (150 mm × 2.1 mm, 3.5 µm particle size) and the MSn spectra were recorded in the negative and positive ion modes. The injection volume was 10 µl. The elution was conducted with water containing 0.1% formic acid (solvent A) and acetonitrile (solvent B). The gradient elution was started at 8% of B and linearly changed to 10% of B in 10 min, then to 25% of B in 30 min and to 98% of B over 10 min, followed by the return to stationary conditions and was re-equilibrated for 10 min. The most important MS parameters were as follow: the ion source ESI voltage −4 kV or 4 kV; nebulization with nitrogen at a pressure of 30 psi at a gas flow rate 9 l/min. Ion source temperature at 310 ºC, skimmer 1: −10 V. The spectra were scanned in the range of 50–3000 m/z.

Second system consisted of UPLC (the Acquity system, Waters, Milford, USA) hyphenated to Q Exactive hybrid MS/MS quadrupole – Orbitrap mass spectrometer. Injection volume was 10 µl. Chromatographic separation for this system were carried out using a water acidified with 0.1% formic acid (solvent A) and acetonitrile (solvent B) with mobile phase flow of 0.4 ml/min in the following gradient: 0–5 min from 10% to 25% B, 5–13 min to 98% B and maintained this conditions for 14.5 min. Up to 15 min system returned to starting conditions and was re-equilibrated for 3 min. Q-Exactive MS operated upon following settings: the HESI ion source voltage −3 kV or 3 kV. The sheath gas flow 48 l/min, auxiliary gas flow 13 l/min, ion source capillary temperature 250 ºC, auxiliary gas heater temperature 380 ºC. The CID MS/MS experiments were performed using collision energy 15 eV. Calibration of this MS/MS in both ionization modes was carried out using calibration solutions: Pierce LTQ Velos ESI Positive ion Calibration Solution and Pierce LTQ Velos ESI Negative ion Calibration Solution (Thermo Scientific). Data were analyzed with Xcalibur version 3.0.63.

The MSn (up to the MS5) and MS/MS spectra were recorded in negative and positive ion modes using a previously published approach (Piasecka et al., 2015, 2017; Ozarowski et al., 2016). The individual compounds were identified via comparison of the exact molecular masses with mean error less than 5 ppm, mass spectra and retention times to those of standard compounds, online available databases (PubChem, ChEBI, Metlin and KNApSAck) and literature data.

Chromatographic data pre-processing

Chromatographic data were analyzed with Empower 2 Chromatography Data Software (Waters, Milford, USA). Normalization by the mass of sample and integration of peaks by ApexTrack Algorithm was done. Additional manual integration events were added in the points of chromatograms where automatic integration did not separate peaks well. Area of each peak expressed as percent of total area of chromatogram were exported in ASCII format and served for statistical analysis. Bar chart that shows the area percent of identified peaks was performed using function ggplot in R package ggplot2.

Venn diagram was implemented in R script using function venn in package gplots.

Cytotoxicity of the extract against human acute lymphoblastic leukemia cell lines

Cell lines

Two human acute lymphoblastic leukemia cell lines were used as the experimental model: CCRF-CEM (ATCC®: CCL-119™) and its multidrug resistant (MDR) variant CCRF-ADR5000 overexpressing ABCB1. MDR cells were obtained by selection with the use of adriamycin from their wild-type counterparts described previously (Paszel et al., 2011).

Viability test

Viability of the human acute lymphoblastic leukemia cells treated with the tested extracts was assessed by the MTT assay described by Mossman (1983). The test was performed as described previously (Paszel et al., 2011). Briefly, CCRF-CEM cells were exposed for 24, 48 and 72 h to the studied extracts in a concentration range of 3.125–100 µg/ml. As a reference compounds four flavonoids: vitexin, isovitexin, apigenin and luteolin were used. CCRF-CEM and CCRF-ADR5000 cell lines were treated with these compounds for 24, 48 and 72 h in the concentration range of 3.125–100 µM. The solvent, methanol in a concentration of 0.25%, was also applied as a control and was identified as not cytotoxic in the used concentration. Experiments were performed in duplicates and were repeated three times. Based on the results from MTT test IC50 values were calculated using CalcuSyn software (BioSoft, Cambridge, UK).

Statistical analysis

All values of phytochemical determinations and results of the study were expressed as means ± SEM. Moreover, results obtained from leukemia cell lines were calculated using T-Student test (p < 0.05).

Results and discussion

Phytochemical studies

Two complementary spectrometric systems: high resolution MS and an ion trap MS enabled to identify eighty two secondary metabolites in crude extracts of Passiflora leaves (Fig. 1; Table 1). Previous study showed, that the highest amount of phenolic compounds (expressed as mg of gallic acid equivalent/g of extract) was measured in P. alata (8.21 ± 0.003 mg/g), P. caerulea (6.23 ± 0.000 mg/g) and P. incarnata (4.85 ± 0.003 mg/g) (Hadaś et al., 2017). Phenolic acid derivatives constituted 18%, 11.5% and 5% of the area of identified peaks of P. alata, P. caerulea and P. incarnata, respectively. The majority of the metabolites belonged to flavonoids. In particular, glycosides of flavonols quercetin and myricetin were found in P. cearulea and P. alata whereas glycosides of flavones apigenin, luteolin and chrysin were the most abundant phytochemicals in all studied plants.

Table 1 Secondary metabolites identified in Passiflora incarnata, P. alata, P. caerulea leaves by HPLC-UV-MS n

No. rt [min] Compound name Fragmentation pathway [m/z] Chemical formula Molecular mass of ions [M-H]- or [M-H]+ Δ ppm λ [nm] P. caerulea P. incarnata P. alata Id l CAS Ref
ESI (-) ESI (+) Calculated Measured
1 3.2 Phenylalanine 166, 120 C9H12NO2 166.086 166.0863 -1.3614 260 * * * 1 63-91-2 Std
2 3.5 Caffeic acid-glucoside 341, 179, 161, 113 C15H18O9 Not detected in HR MS 304sh * 2 Parveen et al., 2013
3 4.3 Pinoresinol 357, 241, 151, C20H21O6 357.13474 357.1344 1.059 275 * 2 487-36-5 Ye et al., 2005; Eklund et al., 2008
4 4.4 Dihydroxybenzoyl-hexoside 315, 153 317, 155 C13H15O9 315.0718 315.0722 -1.1026 281 * * 2 Parveen et al., 2013
5 4.9 Shikimate-hexoside 319, 157 C13H19O9 319.10342 319.1035 -0.111 275, 300 * 3 Parveen et al., 2013
6 5.3 Tryptophan 203, 159, 116 205, 188 C11H13O2N2 205.097 205.0972 -0.9744 285 * * * 1 73-22-3 Std
7 7.1 Dihydroxybenzoyl-pentoside 285, 153 287, 155 C12H13O8 285.0616 285.0616 -0.0215 286 * * * 3 Parveen et al., 2013
8 7.3 Dihydroxybenzoyl-glucosylpentoside 447, 267, 153 C18H23O13 447.11441 447.1144 -0.0078 284 * * 3 Parveen et al., 2013
9 8.8 Luteolin 6,8-di-C-glucoside-7-O-glucoside 771, 651, 609, 489, 399, 369 C33H39O21 771.1978 771.1963 -2.0038 269, 335 * 3 Ferreres et al., 2003
10 10.5 Isovitexin 8-C-arabonoside-7-O-glucoside 725, 635, 605, 563, 473, 443, 353 727, 709, 643, 559 C32H37O19 725.1924 725.1907 -2.3184 270, 332 * * * 3 Ferreres et al., 2003
11 10.5 Quercetin 3'-O-glucoside-7-O-glucosyldeoxyhexoside 771, 609, 301 C33H39O21 771.1978 771.1978 -0.0252 272, 345 * * 3 Kachlicki et al., 2008
12 10.6 Isovitexin 2"-O-glucoside-7-O-glucoside (apigenin 6-C-glucoside 2"-O-glucoside-7-O-glucoside) 755, 593, 455, 413, 293 757, 595, 577, 529, 445, 379, 337, 293 C33H39O20 755.2029 755.1998 -4.1733 269, 332 * 3 Simigriotis et al., 2013
13 11.2 Luteolin 6-C-hydroxybenzoylpentoside-di-O-glucoside 861, 699, 647, 327 C39H41O22 861.21027 861.2095 0.899 269, 340 * 3 Ferreres et al., 2003; Ye et al., 2005
14 11.4 Luteolin 6,8-di-C-glucoside 609, 489, 369, 399 611, 593, 473, 408, 353, 299 C27H29O16 609.1456 609.1477 -3.521 270, 344 * * 2 29428-58-8 Ferreres et al., 2003; Waridel et al., 2001
15 11.9 Apigenin 6,8-di-C-glucoside 593, 473, 383, 353 595, 577, 511, 457 C27H29O16 593.1501 593.1497 -0.7389 269, 333 * * 2 23666-13-9 Ferreres et al., 2003; Waridel et al., 2001
16 12.3 Apigenin 6, 8-di-C-glucoside-7-O-glucoside 755, 593, 473, 383, 353 C33H39O20 755.2029 755.1998 -4.1733 268, 334 * * 3 Ferreres et al., 2003
17 13.5 Luteolin 6-C-pentoside-8-C-glucoside 579, 489, 459, 399, 367 C26H27O15 579.135 579.1376 -2.751 270, 241 * 3 Ferreres et al., 2003
18 14.2 Vitexin 6"-O-glucoside (apigenin 8-C-glucoside 6"-O-glucoside) 593, 473, 431, 311 595, 577, 529, 433, 337, 267, 283 C27H29O15 593.1501 593.1477 -4.0371 271, 335 * 3 639850-16-1 Ferreres et al., 2007; Wojakowska et al., 2013
19 14.6 Isoorientin 7-O-glucoside (luteolin 6-C-glucoside 7-O-glucoside) 609, 489, 447, 327 C27H29O16 609.145 609.1423 -4.3948 272, 345 * * 2 35450-86-3 Ferreres et al., 2007; Simigriotis et al., 2013
20 15.2 Isoorientin (luteolin 6-C-glucoside) 447, 327 449, 431, 383, 353, 299 C21H19O11 447.0922 447.0907 -3.2633 272, 345 * 1 4261-42-1 std
21 15.4 Orientin 7-O-deoxyhexoside (luteolin 8-C-glucoside 7-O-deoxyhexoside) 593, 473, 429, 377, 299 595, 577, 529, 449, 367 C27H29O15 593.1989 593.1479 -3.9288 268, 344 * 3 Ferreres et al., 2007
22 16.1 Apigenin 6,8-di-C-pentoside 533, 473, 443, 413, 383, 353 535, 499, 481, 379, 283 C25H27O13 535.1458 535.1446 2.2345 269, 332 * * 3 Ferreres et al., 2003
23 16.6 Isoscoparin 2"-O-glucoside (chrysoeriol 6-C-glucoside 2"-O-glucoside 623, 443, 323, 309 625, 463, 445, 391, 313 C28H31O16 623.1612 623.1634 -3.5303 243, 271, 349 * 3 97605-25-9 Ferreres et al., 2007
24 17.4 Vitexin 6"-O-pentoside (apigenin 8-C-glucoside 6"-O-pentoside) 563, 473, 443, 413, 341, 311 565, 499, 427, 313 C26H27O10 563.1395 563.1414 3.246 269, 332 * 3 Ferreres et al., 2007
25 17.6 Orientin 6"-O-glucoside (luteolin 8-C-glucoside 6"-O-glucoside) 609, 489, 429, 357, 327 611, 593, 545, 473, 425, 395, 341, 299 C27H29O16 609.1456 609.1477 -3.521 272, 345 * 3 Piasecka et al., 2015; Ferreres et al., 2007
26 18.1 Isovitexin 7-O-glucoside (apigenin 6-C-glucoside 7-O-glucoside) 593, 431, 311 C27H29O15 593.1501 593.1477 -4.0371 269, 335 * 3 20310-89-8 Ferreres et al., 2007
27 18.2 Quercetin glucosyldeoxyhexoside 609, 301 C27H29O16 609.145 609.1424 -4.2946 273, 350 * 3 Cuyckens et al., 2001
28 18.6 Luteolin 7-O-glucosyldeoxyhexoside 593, 447, 413, 285, 257 C27H29O15 593.1501 593.1477 -4.0317 268, 345 * 3 Cuyckens et al., 2001
29 18.8 Apigenin 6-C-pentoside-8-C-glucoside 563, 473, 443, 353, 383 C26H27O14 563.1395 563.1411 3.779 269, 333 * * 3 Ferreres et al., 2003
30 19 Isovitexin 6"-O-deoxyhexoside (apigenin 6-C-glucoside 6"-O-deoxyhexoside) 577, 413, 311 579, 433, 415, 367, 337, 283 C27H29O14 577.15625 577.1563 -0.05 270, 335 * 3 Ferreres et al., 2007
31 19.2 Apigenin 6-C-deoxyhexoside-8-C-pentoside 547, 487, 457, 427, 367, 337, 309, 281 549, 531, 483, 465, 375 C26 H27O13 547.1457 547.1449 -1.488 268, 334 * 3 Ferreres et al., 2003
32 19.6 Isoscoparin 2"-O-deoxyhexoside (chrysoeriol 6-C-glucoside 2"-O-deoxyhexoside) 607, 443, 323, 307 609, 463, 397, 313 C28H31O15 607.1663 607.1697 -2.751 246, 272, 349 * * 3 Ferreres et al., 2007
33 20.8 Apigenin C-glucose C-deoxyhexose 577, 503, 473,443,383, 353 565, 547, 499, 403, 295 C27H29O14 577.1552 577.1533 -3.3312 268, 335 * * 3 Piasecka et al., 2015; Ferreres et al., 2003
34 21 Isovitexin 2"-O-dideoxyhexoside (apigenin 6-C-glucoside 2"-O-dideoxyhexoside) 561, 413, 251 C27H29O13 561.16187 561.1614 0.902 329, 333 * 3 Xu et al., 2013
35 21.1 Vitexin 2"-O-glucoside (apigenin 8-C-glucoside 2"-O-glucoside) 593, 473, 413, 357, 293 595, 577, 457, 283 C27H29O15 593.1501 593.1477 -4.0371 270, 334 * * 3 61360-94-9 Ferreres et al., 2007
36 21.8 Luteolin 6-C-[6"-O-deoxyhexoside]- dideoxyhexoside 561, 327 C27H29O13 561.29169 561.2916 0.071 271, 344 * 3 Ferreres et al., 2007; Xu et al., 2013
37 21.8 Orientin 6"-O-deoxyhexose (luteolin 8-C-glucoside 6"-O-deoxyhexose) 593, 473, 327, 357 C27H29O16 593.1501 593.1497 -0.7389 269, 346 * 3 Ferreres et al., 2007
38 22 Chrysin 6-C-glucoside-8-C-pentoside 547, 457, 427, 337, 309 549, 513, 413, 309 C26H27O13 547.276 547.2766 1.115 264, 330 * 3 Ferreres et al., 2003; Zucolotto et al., 2011
39 23.2 Apigenin 6-C-[2"-O-deoxyhexoside] - pentoside 547, 457, 383, 293 549, 265, 221 C26H27O13 547.276 547.2767 1.334 269, 335 * 3 Ferreres et al., 2007
40 23.4 Luteolin 7-O-[6"-acetyl]-deoxyhexoside 473, 327, 285 C23H21O11 473.10907 473.1089 0.286 269, 346 * 3 Simigriotis et al., 2013
41 24.4 Chrysin 6-C-glucoside 415, 295, 267 C21H19O9 415.1035 415.1037 0.5863 271, 346 * 3 28368-57-2 Zucolotto et al., 2011
42 24.5 Isoorientin 2"-O-glucoside (luteolin 6-C-glucoside 2"-O-glucoside) 609, 489, 429, 357, 327, 309 611, 449, 431, 353, 299 C27H29O16 609.145 609.1424 -4.2946 271, 344 * 3 55196-48-0 Piasecka et al., 2015; Waridel et al., 2001
43 24.9 Chrysin 6-C-[2"-O-glucoside]-glucoside 579, 417, 351 C27H29O14 577.1563 577.1564 0.1618 266, 331 * 3 Wojakowska et al., 2013; Zucolotto et al., 2011
44 25.5 Chrysin 6-C-glucoside-7-O-glucosyldeoxyhexoside 725, 561, 417, 351 C33H39O18 723.2142 723.214 -0.2739 272, 329 * 3 Wojakowska et al., 2013; Zucolotto et al., 2011
45 25.7 Blumenol C-glucoside 373, 211, 193 C19 H15O8 371.0772 371.0772 -0.0018 253 * * 3 135820-80-3 Peipp et al., 1997; Marino et al., 2008
46 25.8 Vitexin (apigenin 8-C-glucoside 433, 367, 337, 309 C21H19O10 431.0978 431.0988 0.001 269, 333 * 1 3681-93-4 std
47 26 Isovitexin (apigenin 6-C-glucoside) 431, 311 433, 415, 367, 283 C21H19O10 431.09833 431.0984 -0.093 269, 334 * 1 38953-85-4 std
48 26.2 Isovitexin 7-O-deoxyhexoside (apigenin 6-C-glucoside 7-O-deoxyhexoside) 577, 413, 311 C27H29O14 577.1563 577.1544 3,2222 269, 335 * 3 Cuyckens et al., 2001
49 26.3 Pinoresinol O-glucosyldeoxyhexoside-O-glucoside 827, 519, 357, 151 C38 H51 O20 827.29913 827.2979 1.466 278 * 3 Eklund et al., 2008
50 26.5 Isovitexin 2"-O-deoxyhexoside (apigenin 6-C-glucoside 2"-O-deoxyhexoside) 577, 413, 293 C27H29O14 577.1563 577.1544 3.2222 269, 335 * * * 3 Zucolotto et al., 2011
51 26.5 Chrysin di-O-glucoside 579, 417, 255 C27H29O14 577.15686 577.1563 1.007 265, 330 * 3 Piasecka et al., 2015; Zucolotto et al., 2011
52 26.6 Apigenin 7-O-glucosyldideoxyhexoside 561, 269 C27H29O13 561.16144 561.1614 0.137 269, 335 * 3 Xu et al., 2013; Wojakowska et al., 2013
53 26.8 Apigenin 7-O-glucosyldeoxyhexoside 577, 269 C27H29O14 577.1563 577.1563 0.779 268, 335 * 3 Kachlicki et al., 2008
54 26.8 Apigenin 6-C-[6"-acetyl-2"-O-deoxyhexoside]-glucoside 619, 559, 455, 293 C29H31O15 619.1668 619.1658 -1.7311 275, 339 * 3 Simigriotis et al., 2013
55 26.9 Quercetin 7-O-glucoside 465, 303 C21H21O12 465.10345 465.1038 -0.858 245, 271, 351 * 1 491-50-9 std
56 27 Scoparin 2"-O-glucoside (chrysoeriol 8-C-glucoside 2"-O-glucoside) 623, 503, 443, 323, 298 C28H31O16 623.1612 623.1636 3.85 242, 269, 348 * 3 124902-15-4 Ferreres et al., 2007; Wojakowska et al., 2013
57 27 Apigenin 7-O-glucosylpentoside 563, 269 C26H27O10 563.1395 563.1414 3.246 269, 335 * 3 Cuyckens et al., 2001
58 27.2 Feruloylquinic acid diglucoside 691, 659, 335, 317, 273 693, 675, 643, 504, 359, 337 C29H39O19 691.2091 691.2079 -1.753 325sh * * 2 Sakalem et al., 2012
59 27.4 Chrysin 6-C-[6", 2"-di-O-deoxyhexoside]-glucoside 707, 453, 277 709, 563, 417, 321 C33H39O17 707.2193 707.2193 0.039 275, 332 * 3 Ferreres et al., 2007; Zucolotto et al., 2011
60 27.6 Luteolin 7-O-glucoside 449, 285 C21H21O11 449.10797 449.1089 -2.148 269, 345 * 1 5373-11-5 std
61 29.6 Chrysin 6-C-[2"-O-pentoside]-glucoside 547, 277, 175 549, 417, 399, 351, 321, 267 C26H27O13 547.276 547.277 1.8979 265, 331 * 3 Ferreres et al., 2007; Zucolotto et al., 2011
62 30.3 Chrysin 6-C-[2"-O-deoxyhexoside]-glucoside 561, 277, 175 563, 417, 399, 351, 321, 267 C27H29O13 561.1614 561.1613 -0.203 269, 331 * * 3 Ferreres et al., 2007; Zucolotto et al., 2011
63 32.7 Blumenol C 211, 175, 133, 119 C13H23O2 211.16896 211.1693 -1.404 255 * 2 36151-02-7 Peipp et al., 1997; Marino et al., 2008
64 34.2 Phillygenin O-glucoside-O-pentoside 665, 503, 371 C32H41O15 665.24664 665.25 2.325 274 * 3 Eklund et al., 2008
65 34.7 Chrysin 6-C-glucoside 417, 351 C21H19O9 415.1035 415.1035 0.0716 265, 333 * 3 28368-57-2 Zucolotto et al., 2011
66 35.4 Isoorientin 2"-O-deoxyhexoside (luteolin 6-C-glucoside 2"-O-deoxyhexoside) 595, 449, 413, 329, 299 C27H29O15 593.1501 593.1522 3.58 269, 346 * 3 Piasecka et al., 2015; Zucolotto et al., 2011
67 36.1 Luteolin O-glucoside-O-deoxyhexoside 595, 449, 431, 287 C27H29O16 593.1507 593.1522 3.58 271, 348 * 3 Kachlicki et al., 2008
68 36.5 Blumenol B glucoside 387, 369, 225, 207 C19H31O8 387.20255 387.2024 0.281 256 * 3 Marino et al., 2008
69 37.4 Apigenin-6-C-deoxyhexoside-7-O-glucoside 577, 415, 311, 283 C27H29O14 577.1552 577.1533 -3.3312 269, 335 * 3 Simigriotis et al., 2013
70 37.5 Blumenol C pentoside 341, 209 C18H29O6 341.19672 341.197 -0.709 255 * 3 Peipp et al., 1997; Marino et al., 2008
71 37.5 orientin 2"-O-dideoxyhexoside (luteolin 8-C-glucoside 2"-O-dideoxyhexoside 579, 431, 413, 335, 299 C27H29O14 577.15668 577.1563 0.695 269, 344 * 3 Piasecka et al., 2015; Xu et al., 2013
72 38.6 Chrysin O-deoxyhexoside-O-glucoside 563, 417, 255 C27H29O13 561.1614 561.1617 0.5668 264, 331 * 3 Wojakowska et al., 2013; Zucolotto et al., 2011
73 39.1 Myricetin glucosyldeoxyhexoside 625, 463, 317 C27H45O16 625.2713 625.2718 0.77 271, 355 * 3 Simigriotis et al., 2013
74 40.1 Cyclopassifloic acid glucoside 697, 535, 517 C37H61O12 697.41718 697.4169 0.473 244 * 3 Yoshikawa et al., 2000
75 41.7 Luteolin 6-C-deoxyhexoside 7-O-glucoside 593, 431, 327 595, 449, 431, 413, 329, 299 C27H29O15 593.1989 593.1479 -3.9288 269, 347 * 3 Zucolotto et al., 2011
76 42.7 Apigenin O-deoxyhexoside-O-glucoside 577, 415, 269 C27H29O14 577.15649 577.1563 0.366 269, 333 * 3 Cuyckens et al., 2001
77 43.6 Dicaffeoylquinic acid 515, 353 517, 193 C25 H39 O11 515.25018 515.2498 0.766 315 * 2 89919-62-0 Clifford et al., 2005
78 44.4 Chrysin 6-C-deoxyhexoside-7-O-glucoside 561, 399, 295 C27H29O13 561.16199 561.1614 1.1106 264, 331 * 3 Zucolotto et al., 2011
79 46 Apigenin 6-C-[2"-O-deoxyhexoside]-deoxyhexoside 561, 397, 293 C27H29O13 561.16144 561.1614 0.1317 269, 335 * 3 Xu et al., 2013; Zucolotto et al., 2011
80 46.7 Chrysin 6-C-glucoside-6"-O-deoxyhexoside- 561, 457, 295 563, 401, 365, 311 C27H29O13 561.16199 561.1614 1.115 266, 333 * 3 Zucolotto et al., 2011
81 47.1 Apigenin-8-C-deoxyhexoside-7-O-glucoside 577, 473, 415, 353, 311, 283 579, 417, 399, 355 C27H29O14 577.15729 577.1563 1.752 269, 334 * 3 Xu et al., 2013
82 47.7 Luteolin 8-C-[6"-O-glucoside]-dideoxyhexoside 577,473, 415, 357, 327 C27H29O14 577.15753 577.1563 2.168 269, 346 * 3 Ferreres et al., 2003; Ferreres et al., 2007; Xu et al., 2013

Legend: aMetabolite identification level according to Metabolomics Standards Initiative recommendation (Sumner et al., 2007). The levels include: 1. Identified compounds; 2. Putatively annotated compounds without chemical reference standards, based upon physicochemical properties and spectral similarity with public spectral libraries; 3. Putatively characterized compound classes based upon characteristic physicochemical properties of a chemical class of compounds, or by spectral similarity to known compounds of a chemical class; 4. Unknown compounds - although unidentified or unclassified these metabolites can still be differentiated and quantified based upon spectral data.Std, identification on the basis of standard compound fragmentation; italic, main peak detected in MS n experiment; ref, references.MW, molecular mass.

Fig. 1 UV chromatograms recorded at 280 nm by UPLC-MS/MS of metabolites detected in extract of: A. Passiflora caerulea; B. P. incarnata; C. P. alata

The diversity of glycosylation site in Passiflora species concerns all identified flavonoids (Fig. 2). O,C-glycosides were in the highest content in all Passiflora species. Interestingly, C-glycosides were observed in high content in P. caerulea (more than 30% of peaks area) and in P. incarnata (more than 20% of peaks area) in set of parameters described in section 2.4. Glucose, deoxyhexose, pentose and dideoxyhexose were found among glycosidic substituents of flavone aglycons. The MS analysis in both negative and positive ionization modes enabled to distinct 6-C- and 8-C-glycosidic bond as well as 2"-O- and 6"-O-glycosides.

Fig. 2 Percentage ratios for groups of identified compounds in the extracts of Passiflora caerulea, P. incarnata, P. alata

In addition, terpenoidal structures such as blumenols B and C (megastigmanes) and cyclopassifloic acid glucoside were also identified (in the group “others” in Fig. 2) in Passiflora species. The highest content of terpenoids was identified in P. alata for which 38% of the area of identified peaks is not the origin of the phenylpropanoid pathway (Fig. 2).

Detailed MS analysis for all detected chemical compounds is presented in Table 1.

Chromatograms of P. caerulea and P. incarnata showed greater similarities in metabolites content than P. alata (Fig. 3). Earlier study (Farag et al., 2016) using hierarchical cluster analysis of Passiflora species showed that they share comparable metabolite profiles. Moreover P. caerulea appeared to be the closed to P. incarnata to terms of overall chemical composition. Five metabolites were common to all species (precursors of phenylpropanoids and terpenoids phenylalanine and tryptophan, as well as dihydroxybenzoyl-pentoside, apigenin 6-C-glucoside 8-C-arabonoside-7-O-glucoside and isovitexin 2"-O-deoxyhexoside). P. alata and P. incarnata had the lower number of common metabolites. Metabolites of P. alata were only partially identified, however the unknown metabolites should not have an impact on similarieties among Passiflora species. The unknown metabolites belonged tentatively to terpenoids rather than phenylpropanoids due to characteristic of UV maximum of absorption and MS fragmentation.

Fig. 3 Venn diagram: similarities in metabolites content between extracts of Passiflora caerulea, P. incarnata, P. alata

The MS analysis in both negative and positive ionization modes enabled to distinct 6-C- and 8-C-glycosidic bond as well as 2"-O- and 6"-O-glycosides. Two different C,O-diglycosides of the flavone luteolin (metabolites 19 and 21, Table 1) are substituted in a different manner. The most abundant product ion at m/z = 447 observed during fragmentation of metabolite 19 in the negative ionization corresponded to the [M−H-162] indicating the presence of an O-glucoside (Figs. A.1A, A.9). An analogous [M−H-146] ion for 21 revealed the substitution with an O-deoxyhexose (Figs. A.1B, A.9). The [Agly+42-H] and the [Agly+72-H] product ions are the same for 19 and 21, however, present in a different ratio. The most abundant [Agly+42-H] ion of 19 indicated the 6-C-glycoconjugate of luteolin (isoorientin), whereas the most abundant [Agly+72-H] ion of 21 referred to the 8-C-glycoconjugate of luteolin (orientin) (Ferreres et al., 2003). On the basis of the above information metabolite 19 and 21 were identified as isoorientin 7-O-glucoside and orientin 7-O-deoxyhexoside, respectively.

Apigenin substituted with deoxyhexose and glucose was identified in six isoforms: compounds 30, 33, 50, 53, 69 and 76. Analysis in negative ionization enabled to distinguished compounds 30 and 50 on the basis of their main product ions in MS2 and MS3 according to Piasecka et al. (2015). The first mentioned compound was characterized by the major product [Agly+42-H] ion typical for C-glycosides of flavones and the deprotonated [M−H-164] and [M−H-266] ions indicated on deoxyhexose substituted to glycosidic carbon (Figs. A.2A, A.9). The only one possible place of substitution with such a fragmentation is C-[6"-O-deoxyhexosyl]-glucosides. The former compound has the major product [Agly+(42–18)-H] ion characteristic for structure of the C-[2"-O-deoxyhexosyl]-glucosides of flavones (Figs. A.2B, A.9). The deprotonated [M−H-164] ion confirmed that deoxyhexose is substituted to glycosidic moiety. Thus, 30 and 50 were identified as isovitexin 6"-O-deoxyhexoside and isovitexin 2"-O-deoxyhexoside, respectively. Detachment of fragments 74, 104, 90 and 120 amu as well as the major product [Agly+84-H] ion at m/z = 353 of isomeric 33 indicated on structure of di-C-glycosides of flavone according to Ferreres et al. (2003) (Figs. A.2C, A.9). The intensity of deprotonated ions formed after detachment of glucose and deoxyhexose moiety have similar intensity, thus the distinction of particular substituents between 6-C and 8-C of aglycon moiety was rather problematic. Therefore, 33 was tentatively identified as apigenin C-glucose C-deoxyhexose. Metabolites 53 and 76 have the major deprotonated product ion at m/z = 269 similar to apigenin standard and therefore indicated on O-type glycosylation on flavone skeleton (Figs. A.2D and E, A.9). The differences in fragmentation pattern in MS2 and MS3 between compounds 53 and 76 enabled to distinguish their glycosidic patterns. In 53 the [M−H-308] ion indicated on O-glucosyldeoxyhexoside substituent according to Kachlicki et al. (2008). Proportionally high intensive [M−H-164] ion indicated on deoxyhexosyl(1→2)glucosidic bond. In 76 a high intensive [M−H-162] and [M−H-162-146] ions reflected successive detachment of glucose and deoxyhexose which suggested substitution of both glycosides on separate hydroxyl groups on aglycon (Cuyckens et al., 2001) (Figs. A.2E, A.9). Thus, 76 was identified as apigenin O-deoxyhexoside-O-glucoside. Compound 69 represented another isomeric structure of apigenin deoxyhexosylglycoside on the basis of the major product [Agly+42-H] ion. The high intensive [M−H-162] ion indicated on O-glucose substituted to aromatic ring of aglycon and [M−H-162-104] ion indicated on C-deoxyhexose (Ferreres et al., 2007) (Figs. A.2F, A.9). Therefore 69 was assumed as apigenin 6-C-deoxyhexoside 7-O-glucoside. Metabolites 36, 52, 71 and 82 were identified as flavone structures with poorly known dideoxyhexosides. Similar structures were previously isolated and identified as 8-C-β-digitopyranoside and 8-C-β-boivinopyranoside of luteolin and apigenin by NMR from P. edulis leaves and stems (Xu et al., 2013) (for P. edulis, P. incarnata is recognized as synonym). The main product ion at m/z = 327 in negative ionization mode of 36 was typical for C-glycosides of luteolin as described Ferreres et al. (2007) (Fig. A.3A). The most abundant [Agly+42-H] ion indicated on 6-C-glycosides rather than 8-C isomer. The [M−H] ion of compound 36 showed losses of 234 amu (88 + 146) reflecting the structure of 6-C-dideoxyhexose (1→6)deoxyhexose. Precise defining of certain glycoside residue reacquires further NMR analysis since three isomeric deoxyhexosides: rhamnose, fucose and chinovose have been reported in Passiflora edulis fo. flavicarpa (Li et al., 2011) (Chinovose is isomer of rhamnose and is defines as isorhamnose or 6-deoxy-α-D-glucopyranose). Therefore, compound 36 was identified as luteolin 6-C-[6"-O-deoxyhexoside]-dideoxyhexoside. The fragmentation of deprotonated compound 52 yielded losses of 292 amu (162 + 130) derived from the glucosyl-dideoxyhexoside moiety giving the [Agly-H] product ion, typical for apigenin. The place of glycosidic substitution was determined as 7-OH at aglycon moiety on the basis of similarities in fragmentation scheme with other flavone derivatives described in literature (Piasecka et al., 2015; Ferreres et al., 2007). Thus, the diglycosides was identified as apigenin 7-O-glucosyldideoxyhexoside. Compounds 66 and 71 was detected only in positive ionization in which protonated [M+H-146]+ and [M+H-130]+ ions of compounds 66 and 71, respectively followed by fragmentation adequate for C-glucoside allowed to assume that both compounds have structure of 6-C-[2"-O-glycoside]-glucoside of luteolin according to Piasecka et al. (2015). Thus, 66 was identified as isoorientin 2"-O-deoxyhexoside and 71 as isoorientin 2"-O-dideoxyhexoside. Further investigation is necessary to study diversity of O- and C-dideoxyhexosyl substituents in Passiflora species. Dideoxyhexose in structure of 82 were identified as C-linked to aglycon moiety, because glucose fragment 162 amu corresponding to entire glucose moiety and main [Agly+42-H] product ion was similar to 23 (Fig. A.3B). Glucosides and pentosides of phenolic acids such as dihydroxybenzoic and shikimic acids (2, 5 and 7) were observed in lesser proportion. These compounds have been tentatively identified to be phenolic acids glucosides and not the respective esters on the basis of the MS fragmentation pattern. Losses of 162 amu constitute a typical fragmentation pattern of the glucose moiety of compounds 4, 5, as well as losses of 132 amu adequate for pentose moiety in 7 in the negative ion mode. In addition, glucoside of blumenol C (45), a megastigmane terpenoid widely distributed in plant kingdom (Peipp et al., 1997) was also detected. Their UV maximum absorbance is about 250 nm, thus presence chromatographic peak for the compound in 280 nm indicated on relative high amount of the compound. The fragmentation of 45 in both ionization modes is similar to fragmentation of hydroxyferulic acid glucoside (Piasecka et al., 2015) (Fig. A.4A) which can lead to misidentification of the isobaric structures. Nevertheless, determination of accurate masses of 45 in high resolution mass spectrometer confirmed the structure of blumenol C glucoside (Fig. A.4B) which is identified for the first time in Passiflora species.

Cytotoxicity of the extracts and chemical compounds

Several studies showed that flavonoids can play important beneficial roles in chemoprevention and the usage of plant-derived natural compounds is a promising approach for the therapy of malignant disorders via various mechanism of pharmacological action (Sak, 2014). Today, many flavonoids are known to exert anticancer potential (e.g. antileukemic activity) both in in vitro and in vivo models (Caxito et al., 2015), for example apigenin, luteolin, quercetin, chrysin, myricetin. According to Moghaddam et al. (2012) the hydroxyflavones (luteolin, apigenin) exerted comparable antiproliferative activities against malignant cells. Although it was previously reported that the extract of P. incarnata (synonym of P. edulis), the most important medicinal plant from Passifloraceae, possesses cytotoxic activity against cancer cells (Ehrlich Ascites Carcinoma) (Sujana et al., 2012), however still very little is known about the antitumor/antileukemic potential of extracts from plants belonging to the genus Passiflora. This prompted us to assess the cytotoxic potential of crude extracts of P. incarnata, P. alata and P. caerulea leaves against human leukemic cells. Due to the fact, that the multidrug resistance of neoplastic cells is a phenomenon which is one of the most important causes of chemotherapy failure in malignant diseases (Paszel et al., 2011), we decided to investigate the cytotoxic activity of the tested extract in multidrug resistant ABCB1 expressing – CCRF-ADR5000 leukemia cells.

Results showed that the most potent against human acute lymphoblastic leukemia CCRF-CEM cells was extract from P. alata leaves with the IC50 value of 91.2 µg/ml after 72 h of treatment (Table 2). Calculation of IC50 factor for the shorter exposure times (24 and 48 h) was not possible, due to the low activity of the extract.

Table 2 IC50 values obtained for CCRF-CEM cells treated with crude extracts of Passiflora incarnata and P. alata during 24, 48 and 72 h. 

IC50 values ± SD (µg/ml)
CCRF-CEM
24 h 48 h 72 h
Extract of P. incarnata leaves nd nd nd
Extract of P. alata leaves nd nd 91.2 ± 1.1

nd, not detected.

After 72 h of treatment with 100 µg/ml of P. alata leaves extract we observed a 60% growth inhibition of CCRF-CEM cells (p < 0.001) (Fig. 4). These results may correspond to our previous study in which it was shown that P. alata leaves extract contained the highest concentration of phenolic compounds (Hadaś et al., 2017). Moreover, quantitative analysis (HPLC-DAD) showed that this extract contained the highest level of apigenin (9.51 mg/100 g dry weight of extract) in comparison with extracts of P. caerulea and P. incarnata (Hadaś et al., 2017). In addition, the biological activity of P. alata extract can be explained by the highest content of terpenoids. The lower concentrations of the extract were significantly less active. In shorter treatment times we did not detect any significant cell viability reduction. Extract of P. incarnata leaves was less potent against CCRF-CEM cells and the highest concentration (100 µg/ml) induced only a 25% inhibition of cells growth (p < 0.01) (Fig. 5), whereas the extract from leaves of P. caerulea did not show any statistically significant effect (data not shown). Moreover none of the studied extracts were active against multidrug resistant CCRF-ADR5000 cells. Probably the lack of activity in multidrug resistant cells may be the result of the removal of its active components into the extracellular environment by transmembrane protein ABCB1 (data not shown).

Fig. 4 Influence of the extract of Passiflora alata on leukemic cells (CCRF-CEM). Legend: CCRF-CEM was treated with different concentrations of the extract (concentration range of 3.125–100 µg/ml for 72 h. Each point represents the mean ± SD of four independent experiments performed in duplicate (***p < 0.01). 

Fig. 5 Influence of the extract of Passiflora incarnata on leukemic cells (CCRF-CEM). Legend: CCRF-CEM was treated with different concentrations of the extract (concentration range of 3.125–100 µg/ml for 72 h). Each point represents the mean ± SD of four independent experiments performed in duplicate (**p < 0.01). 

Because the most active was P. alata extract containing apigenin and luteolin, in this study tests with using pure compounds were carried out. It was observed that apigenin showed cytotoxic activity in concentration range of 50–100 µM against CCRF-CEM and CCRF-ADR5000 cells (Figs. A.5, A.6) with the IC50 value of 99.6 µM and 68.7 µM after 72 h of treatment, respectively (Table 3). Apigenin was more potent against multidrug resistance CCRF-ADR5000 cells after 48 and 72 h of treatment (Fig. A.6). Compound used in the concentration of 100 µM reduced the population of viable CCRF-ADR5000 cells to 36% after 48 h and to 23% comparing to untreated control cells. Luteolin contained in P. alata extract in concentration of 0.78 mg/100 g dry weight of extract (Hadaś et al., 2017) was the most active chemical compound (Figs. A.7, A.8). It showed a significant cytotoxic activity against CCRF-ADR5000 cells in the whole range of concentrations. Even the lowest concentration of the compound (3.125 µM) caused a significant decrease in CCRF-ADR5000 cells viability. Observed effect was time and dose dependent and after 72 h of treatment with apigenin (100 µM) the population of viable CCRF-ADR5000 cells was reduced to 4%. Values of IC50 factor obtained for multidrug resistant CCRF-ADR5000 cells treated with luteolin were significantly lower comparing to IC50 calculated for wild-type cells (Table 3). This observation indicates the multidrug resistance reduction potential of the compound in the tested leukemic cells. Furthermore, vitexin and isovitexin (contained in passiflora extracts) did not exert any effect in both cell lines (data not shown). Viability of the cells treated with these compounds was not reduced within 72 h of treatment.

Table 3 IC50 values obtained for CCRF-CEM and CCRF-ADR5000 cells treated with apigenin and luteolin during 24, 48 and 72 h. 

IC50 values ± SD (µM)
CCRF-CEM CCRF-ADR5000
24 h 48 h 72 h 24 h 48 h 72 h
Apigenin nd 101.2 ± 2.1 99.6 ± 0.8 nd 83.4 ± 1.4a 68.7 ± 1.7a
Luteolin 91.2 ± 1.1 78.2 ± 1.4 49.4 ± 0.5 84.2 ± 0.9a 65.1 ± 0.7a 25.5 ± 0.5a

nd, not detected.

ap< 0.001 comparison between both cell lines in the same time point.

Conclusions

Our studies showed that crude extracts from leaves of P. alata showed the most potent and statistically significant viability reduction activity against human acute lymphoblastic leukemia CCRF-CEM. However, the crude extract of P. incarnata (synonym of P. edulis) showed only poor activity and crude extract of P. caerulea leaves did not exert any effect. Our results of phytochemical analysis, similarly as Farag et al. (2016), suggest that P. caerulea may be taken into account as a substitute of the P. incarnata, although our pharmacological studies indicated that activity of P. caerulea extract differs from the extract P. incarnata. Thus, despite similarities in quality phytochemical profile, quantity differences in chemical compounds between two extracts may determine their pharmacological (antileukemic) potency. The highest activity of P. alata extract can be related to the highest content of phenolic compounds, terpenoids, and also apigenin and luteolin. Summarizing, the crude extract from P. alata leaves may be considered as a substance for complementary therapy for cancer patients.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bjp.2018.01.006.

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Received: October 19, 2017; Accepted: January 23, 2018; Published: March 10, 2018

* Corresponding author. mozarow@ump.edu.pl

Conflicts of interest

The authors declare no conflicts of interest.

Authors contributions

MO has made substantial contribution to conception and design and carried out the coordination of the research, acquisition, analysis and interpretation of data; made plant extracts and participated in the phytochemical analysis (HPLC-ESI-MSn, UPLC-PDA). Moreover MO carried out the preparation of the manuscript and is a corresponding author. AP, PK carried out the phytochemical investigation (HPLC-ESI-MSn, UPLC-PDA). DCh has been involved in drafting the manuscript and performed critical analysis of material and methods, results and discussion; drew the chemical structures of selected compounds. AG participated in interpretation of phytochemical data. AJP, AR, MR carried out the antileukemic activity studies. AS carried out bar chart and Venn diagram. PM performed revising it critically for important intellectual content and have given final approval of the version to be published. ASM, AK performed the bibliographic data collection. BT was responsible for identifying the plant material and correcting the manuscript.

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